Mass spectrometry (MS) is a widely accepted analytical technique in the chemical sciences to obtain qualitative and quantitative information from a sample. MS is commonly used to determine molecular weight, identify chemical structures, and accurately determine the composition of mixtures. MS is becoming increasingly important in biological research to determine the structure of organic molecules based on the fragmentation pattern of ions formed when sample molecules are ionized.
A technique for focusing and declustering trace ions travelling into a vacuum chamber is disclosed in U.S. Pat. No. 4,121,099. A tandem quadrupole-based mass spectrometer for selected ion fragmentation studies is disclosed in U.S. Pat. No. 4,234,791. A technique for analyzing chemical compounds involving the formation of fine spray droplets of a solution containing the compound to be detected and electronically charging the spray droplets such that ionized molecules of interest is disclosed in U.S. Pat. No. 4,300,044. Techniques for vaporizing liquid solutions in order to detect physical or chemical properties of the samples are disclosed in U.S. Pat. Nos. 4,730,111 and 4,814,612. Relevant technical articles include Mann et al., "Interpreting Mass Spectra of Multiply Charged Ions," Anal. Chem., Vol. 61, pp. 1702-1708 (1989); and Loo et al., "Peptide and Protein Analysis by Electrospray Ionization-Mass Spectrometry and Capillary Electrophoresis-Mass Spectrometry," Analytical Biochemistry, Vol. 179, pp. 404-412 (1989).
Electrospray ionization is similar in some respects to thermospray techniques. U.S. Pat. No. 4,883,958 discloses an improved interface for coupling liquid chromatography to solid or gas phase detectors involving thermospray vaporization. An improved thermospray vaporizer, and more particularly a vaporizer probe employing a reduced diameter nozzle, is disclosed in U.S. Pat. No. 4,902,891.
U.S. Pat. No. 4,999,493 and the references cited therein also disclose electrospray ionization techniques. Electrospray droplets are passed into an ion generating chamber that is maintained at a selected pressure. The walls of the ion-generating chamber are controllably heated at a temperature that will desolvate the droplets and produce ionized molecules of interest for analysis by the mass spectrometer. Electrospray (ES) ionization mass spectrometry is becoming widely used in the analysis of biological molecules, especially peptides and proteins. Continuous flow ionization has the capability of accurately measuring the masses of large proteins as well as small peptides and other organic molecules. ES can operate under variable solvent conditions, which allows the technique to be used as an interface for coupling various separation techniques, such as liquid chromatography (LC), to MS. Reverse phase LC can also be used to effectively desalt and concentrate a sample prior to its introduction into the mass spectrometer, which may be essential to the success of the ES analysis.
One of the most challenging applications of modem analytical technology in biological research requires the analysis of compounds such as endogenous metabolites and hormones at low femtomole concentrations and in microliter volumes of the solutions that contain relatively high salt concentrations. MS can be successfully employed on such samples and can provide analytical capabilities at the molecular level with high-mass measurement accuracy. The specificity of MS is further enhanced in the tandem MS mode. MS has proven to be essential in the neurosciences for monitoring physiologically active substances, such as neuropeptides. The majority of neuropeptide release studies have been performed by radioimmunoassay (RIA) or radioreceptorassay (RRA), which have desired sensitivity but lack specificity due to antibody-reactivity.
MS has been used to identify neuropeptides isolated from pooled brain tissues. After extensive chromatographic purification procedures, samples have been analyzed by fast-atom bombardment tandem MS with a detection sensitivity of approximately 50 pmol. Constant-infusion ES analysis of proteins has resulted in sensitivities in the range of 200 fmol/.mu.L to 1 pmol/.mu.L. ES ionization coupled to either LC or capillary zone electrophoresis (CZE) has greatly increased sensitivity for both peptides and proteins. Projection limits for peptides from LC-ES procedures are typically in the 100 to 500 fmol range. CZE-ES results show attomole sensitivity for the total amounts of analyte loaded, although because of low load volumes, sample solutions have to be relatively highly concentrated. Biological extracellular concentrations of neurosubstances are typically 4 to 6 orders of magnitude lower at the low attomole/.mu.L level.
Various techniques are designed to accommodate less sample material. These techniques typically have high sensitivity but require loading of high concentration samples, which often are not readily available. An article by Gale et al. entitled "Small Volume and Low Flow-rate Electrospray Ionization Mass Spectrometry of Aqueous Samples," Rapid Communications in Mass Spectrometry, Vol. 7, pp. 1017-1021 (1993), discusses using proposals for accommodating low flow rate electrospray ionization analysis.
Improved techniques are thus required to take full advantage of electrospray capabilities, particularly for those instances where ultrahigh sensitivity is required or where the sample of interest from the chromatograph has a relatively low flow rate. The disadvantages of the prior art are overcome by the present invention, and improved techniques are hereinafter disclosed utilizing electrospray for improved ionization of molecules of interest to enhance the reliability and sensitivity of the mass spectrometric or other gas-phase analysis.